Calicheamicin g1I (CLM), is a member of the structurally unprecedented and growing family of diynene antitumor antibiotics. Reductive activation by thiols and/or simple thermal rearrangement generates diradical species by an electrocyclization process that is characteristic of this class. When bound in the minor groove of DNA, these radicals initiate helix cleavage by hydrogen abstraction from one or both strands. The resulting DNA radicals react with molecular oxygen and fragmentation ensues. While single-strand breaks are typically the major cleavage event observed, CLM is both notably sequence-selective and very largely a double-strand cutter. Using a diverse array of experiments we seek to understand the origins of these properties and how CLM can be used as a probe of protein/nucleic acid structure and in the design of useful tools in molecular biology. Extensive experiments are planned with nucleosomes to study the occurrence of "hot spots" to understand whether CLM cleavage is associated with physical location in the nucleosome or features of particular DNA sequences. Automated methods will be used to evaluate large amounts of data generated in mixed sequence nucleosomes. A degradation product of CLM having essentially no sequence selectivity but retaining double-strand cutting properties will be linked to homeodomains to achieve specific binding for high efficiency cleavage of DNA. Success in this effort will be evaluated in tests with DNA sequences already in hand and will lay the groundwork for eventual experiments to be undertaken for gene identification e.g. in Drosophila. Finally, CLM reaction with transfer-, ribosomal- and messenger-RNA will be investigated. In eukariotic cells, RNA is a more accessible target than DNA. These experiments will monitor the susceptibility of RNA to reaction with CLM and particularly with rRNA in the ribosome for comparison to nucleosomes and chromatin. Calorimetry has been used to measure, by ITC and CD titration experiments, the association of CLM with a specific dodecamer containing a strong binding site.